ИСТИНА

Ribosome is a molecular machine for protein biosynthesis. Inactivation of ribosome in the course of translation may happen due to a set of different factors, including antibiotics, chemical modifications, and damage by ribotoxins. One of the mechanisms of the cellular response to the accumulation of non-functional ribosomes is the activation of their degradation by a specific form of autophagy called ribophagy. Only two specific ribophagy receptors have been described in the literature: the NUFIP1 protein, which directly interacts with the ribosome under starvation conditions, and the large ribosome subunit protein RPL12, which serves as a conserved ribophagy receptor in many organisms. Since the complete mechanism of ribophagy is not fully understood, the search for genes involved in this process is an urgent task. To do this, we performed a genome-wide CRISPR/Cas9-based screening using a fluorescent ribophagy reporter, which is a cell line with ribosomes labeled by RPL29 with two fluorescent proteins, mCherry and GFP. When ribophagy is activated, the ratio of mCherry to GFP fluorescence is increased due to GFP fluorescence quenching in the acidic pH of autophagolysosomes. Using genome-wide Brunello library, we identified the most promising targets for further study, including the ubiquitin ligase TRIM25, for which we have shown specific binding to the ribosome under ribophagy induction, leading to ribosomal ubiquitination and degradation.